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Phosphorylation of replication protein A and its significance in the cellular response to DNA damage.

机译:复制蛋白A的磷酸化及其在细胞对DNA损伤的反应中的意义。

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摘要

Post-translational modification of proteins provides a cellular mechanism to switch on or off many diverse cellular processes. In response to slowed or stalled replication forks, which is defined as replication stress, phosphorylation enables rapid activation of proteins involved in DNA replication, repair, and cell cycle checkpoint signaling. One component of the replication stress response is Replication Protein A (RPA), a heterotrimeric protein with a middle subunit (RPA2) that becomes phosphorylated at multiple sites in a cell cycle-dependent as well as in a damage-dependent manner. Damage-induced hyperphosphorylation of RPA has been implicated to interfere with replication and to be involved in checkpoint signaling. This dissertation was undertaken to study the role of RPA phosphorylation in response to replication stress. Using Nijmegen breakage syndrome 1 (NBS1) cells stably transfected with vectors expressing phosphomutant forms of Nbs1, as well as HeLa cells transiently transfected with antibodies specific for the Nbs1 phosphorylation sites we found that hydroxyurea (HU)-induced RPA phosphorylation required ATR-dependent phosphorylation of NBS1. Interference with RPA hyperphosphorylation delayed HU-induced apoptosis.;We further demonstrated that RPA hyperphosphorylation was impaired in various head and neck squamous cell carcinoma (HNSCC) cell lines that displayed increased sensitivities to cisplatin and etoposide. Cisplatin-sensitive HNSCC that had been rendered more resistant to cisplatin by repeated exposure to cisplatin phosphorylated RPA2 in response to cisplatin and etoposide treatment. To investigate the consequences of impaired damage-induced RPA2 phosphorylation, we generated HNSCC cells stably expressing a phosphomutant RPA2 with ten phosphorylation sites mutated to alanines. The increased sensitivity to cisplatin treatment was paralleled by a defect in the recovery from cisplatin-induced replication slow-down and persistent replication stress. RPA2 mutant cells only displayed a minor increase in sensitivity to UV-C but showed decreased binding of RPA to ERCC1/XPF, an endonuclease implicated in crosslink repair and Nucleotide Excision Repair (NER). These data provide evidence for a critical role of damage-dependent RPA2 phosphorylation in the cellular response to replication stress, and suggest a requirement for phosphorylated RPA in crosslink repair that is distinct from its function in NER.
机译:蛋白质的翻译后修饰提供了开启或关闭许多不同细胞过程的细胞机制。响应于缓慢或停滞的复制叉(定义为复制压力),磷酸化可快速激活涉及DNA复制,修复和细胞周期检查点信号传导的蛋白质。复制应激反应的一个组成部分是复制蛋白A(RPA),它是具有中间亚基(RPA2)的异三聚体蛋白,在细胞周期依赖性和损伤依赖性的多个位点都被磷酸化。 RPA的损伤诱导的过度磷酸化被认为会干扰复制,并参与检查点信号传导。本文旨在研究RPA磷酸化在复制应激中的作用。使用表达了Nbs1的磷酸突变体的载体稳定转染的奈梅亨断裂综合症1(NBS1)细胞以及用Nbs1磷酸化位点特异性抗体瞬时转染的HeLa细胞,我们发现羟基脲(HU)诱导的RPA磷酸化需要ATR依赖的磷酸化NBS1。干扰RPA过度磷酸化可延缓HU诱导的细胞凋亡。我们进一步证明,RPA过度磷酸化在表现出对顺铂和依托泊苷敏感性增强的各种头颈鳞状细胞癌(HNSCC)细胞系中受损。通过对顺铂和依托泊苷治疗的反复暴露于顺铂磷酸化的RPA2,已使顺铂敏感的HNSCC对顺铂具有更强的抵抗力。为了研究损伤诱导的RPA2磷酸化受损的后果,我们生成了稳定表达具有10个磷酸化位点突变为丙氨酸的磷酸突变型RPA2的HNSCC细胞。对顺铂治疗的敏感性增加与顺铂诱导的复制减慢和持续复制压力的恢复缺陷有关。 RPA2突变细胞仅显示出对UV-C的敏感性略有增加,但显示RPA与ERCC1 / XPF的结合减少,ERCC1 / XPF是一种涉及交联修复和核苷酸切除修复(NER)的核酸内切酶。这些数据提供了证据证明损伤依赖性RPA2磷酸化在细胞对复制压力的反应中起着关键作用,并提出了在交联修复中需要磷酸化RPA的要求,这与其在NER中的功能不同。

著录项

  • 作者

    Manthey, Karoline C.;

  • 作者单位

    University of Nebraska Medical Center.;

  • 授予单位 University of Nebraska Medical Center.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 150 p.
  • 总页数 150
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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